PROJECT SUMMARY Staphylococcus aureus is one of the most problematic bacterial pathogens in our healthcare settings. S. aureus can survive and persist in the host by developing into an encased community of cells called a biofilm, and numerous studies have demonstrated that biofilms are resistant to host immune defenses and chemotherapies. Our central PPG hypothesis is that S. aureus biofilm development creates unique metabolic niches that promote an immune suppressive environment. In this proposal (Project 3), we are focusing on the contribution of S. aureus extracellular enzymes to biofilm growth and maturation, persistence in the host, and ultimately dissemination to a new site. Of the many enzymes that S. aureus secretes, we will prioritize hyaluronidase (HysA) and nuclease (Nuc1), as they have commonalities in biofilm-host interaction phenotypes and regulatory schemes. We recently demonstrated that hyaluronan accumulates in an S. aureus biofilm infection and that HysA can degrade this host glycosaminoglycan to disaccharides (HA-DS). Our preliminary studies indicate that HA-DS can serve as a carbon source through an unknown catabolic pathway, and this disaccharide has additional anti-inflammatory properties that could be contributing to the persistent nature of S. aureus biofilm infections. In Specific Aim 1, we will determine the role and regulation of hyaluronan metabolism in S. aureus biofilm maturation. We will characterize the HA-DS catabolic pathway using genetic analysis and labeling studies in collaboration with the Metabolomics Core. We will also test catabolic pathway knockouts in biofilm maturation and foreign-body infections, and investigate the contribution of CodY and CcpA to regulation of hyaluronan catabolism. Additionally, S. aureus will be grown on HA-DS and RNAseq performed to identify global transcriptomic changes. In Specific Aim 2, we will investigate how S. aureus enzymatic degradation of host polymers impacts the biofilm anti-inflammatory state. In collaboration with Dr. Tammy Kielian (PPG Project 4), we will assess the effect of HA-DS, as well as Nuc1 and HysA enzymes and their regulators, on immune cell function. We will also determine the impact of HA-DS and HysA inhibitors on biofilm infection, and test whether HA-DS is a biomarker for S. aureus in human synovial fluid from patients with prosthetic joint infection (PJI). In Specific Aim 3, we will examine S. aureus exo-enzyme regulation and function in biofilm dispersal. We hypothesize that CodY controls dissemination from S. aureus biofilms in an enzyme and nutrient dependent manner. We will investigate the contribution of Nuc1 and HysA, along with CodY and SaeRS regulators, to biofilm dispersal in vitro and during foreign body infection. We will also examine the impact of nutritional status on CodY activity during biofilm formation and dispersal in collaboration with Dr. Ken Bayles (PPG Project 1) and the Bioimaging Core. Finally, we will determine the role of aureusimine molecules in S. aureus biofilm development. Collectively these studies will define the contribution of S. aureus exo-enzymes to biofilm metabolism, development and persistence, potentially leading to innovative therapies for biofilm infections.